Rubella virus (RUB) is a major human pathogen. RUB infection during pregnancy can lead to severe birth defects (congenital rubella syndrome or CRS). Rubella is controlled in the U.S. by use of live, attenuated vaccines. However, two challenges confront the vaccination program. First, the virus is endemic in much of the world, necessitating a continuing vigilant vaccination effort to prevent reintroduction as occurred between 1989 and 1991. Secondly, in adult women, the vaccine has been associated with chronic arthritis thought to be due to the persistence of the vaccine virus. Another important medical aspect of RUB is that CRS patients suffer from a high incidence of diabetes, providing the best association of a specific human virus with an autoimmune disease. The long-term goal of the proposed research is a thorough study of the molecular biology of RUB. Despite its medical importance, progress on the molecular biology of RUB has been slow, primarily because RUB replicates slowly and to low titers in cell culture. A recent landmark event in the molecular biology of RUB was development of an "infectious clone". An infectious clone is a complete cDNA copy of an RNA virus genome contained in a plasmid from which infectious RNA transcripts can be synthesized in vitro. The first specific aim of the proposed research is to use the infectious clone for two ends: 1). to study by site-directed mutagenesis the function of specific regions of the genome and the encoded proteins, and 2). to develop an infectious clone based on the live, attenuated vaccine virus. The vaccine infectious clone will be introduced into a "genetic immunization" vector, a plasmid from which the genome RNA will be expressed in cells transfected with the recombinant plasmid alone, initiating virus replication. This recombinant vector could be the basis of a DNA vaccine which could be used in worldwide vaccination programs against RUB. The vaccine infectious clone could also be used to modify the vaccine to avert the complications encountered in adult women. The second specific aim is to study the protease responsible for processing the virus replicase or nonstructural proteins (NSPs). As part of this effort, the NSP open reading frame (ORF1) of hepatitis E virus (HEV) will be expressed to analyze its processing. Computer alignment has revealed that the NSP-ORFs of RUB and HEV are more closely related to each other than to other viruses, despite the disparity in structure of RUB and HEV. HEV is the major cause of enterically transmitted non-A, non-B hepatitis and is associated with a high mortality rate in pregnant women. Study of the NSP proteases of RUB and HEV could lead to design of antiviral drugs against both viruses. The third specific aim of the proposed research is to study the three dimensional structure of both the RUB capsid protein by X-ray crystallography and of the RUB virion by cryo-electron microscopy in collaboration with scientists at Purdue University who specialize in structural biology.